Synthesis, DFT Study, and Antitumor Activity of Some New Heterocyclic Compounds Incorporating Isoxazole Moiety

Article abstract of DOI:10.1002/jccs.201700064

Thiazolidin-4-one derivative 3 was synthesized by the transformation of chloroacetamide derivative 2 with NH₄SCN.The condensation of 3 with p-anisaldehyde afforded the corresponding arylidene derivative 4. Also, the alkylation of chloroacetamide derivative 2 with different heterocyclic compounds was investigated. Annulation of 5-amino-3-methylisoxazole (1) with α-halocarbonyl compounds 12 and 14 furnished pyrrolo[3,2-d]isoxazole and isoxazolo[5,4-b]azepin-6-one derivatives 13 and 15, respectively, while reaction of 1 with 1-chloro-4-(chloromethyl)benzene gave the monoalkylated product 17. The newly synthesized compounds were screened for their antitumor activity, and the geometry optimizations are in a good agreement with the experimentally observed data.

Full text of DOI:10.1002/jccs.201700064

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis, DFT Study, and Antitumor Activity of Some New Heterocyclic Compounds
Incorporating Isoxazole Moiety
*
Wafaa S. Hamama, Mona E. Ibrahim and Hanafi H. Zoorob
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt
(Received: February 24, 2017; Accepted: July 6, 2017; DOI: 10.1002/jccs.201700064)
Thiazolidin-4-one derivative 3 was synthesized by the transformation of chloroacetamide deriva-
tive 2 with NH4SCN.The condensation of 3 with p-anisaldehyde afforded the corresponding aryli-
dene derivative 4. Also, the alkylation of chloroacetamide derivative 2 with different heterocyclic
compounds was investigated. Annulation of 5-amino-3-methylisoxazole (1) with α-halocarbonyl
compounds 12 and 14 furnished pyrrolo[3,2-d]isoxazole and isoxazolo[5,4-b]azepin-6-one deriva-
tives 13 and 15, respectively, while reaction of 1 with 1-chloro-4-(chloromethyl)benzene gave the
monoalkylated product 17. The newly synthesized compounds were screened for their antitumor
activity, and the geometry optimizations are in a good agreement with the experimentally
observed data.
Keywords: Isoxazole; Chloroacetamide; Thiazolidin-4-one; Alkylation; In vitro antitumor activity.
INTRODUCTION
There are numerous biologically active molecules
with adjuvant arthritis.16 Thiazolidin-4-one derivatives
are important compounds because of their broad
range of biological activities17–23 such as antifungal,
antitumor, antibacterial, antitubercular, anticonvul-
sant, antihistaminic, anti-inflammatory, antiviral, and
cardiovascular.24–27 Isoxazoles functionalized with an
additional nitrogen-containing group have found
many applications;28a thus it was considered of value
to synthesize some new heterocyclic derivatives incor-
porating two active moieties in a single molecular
framework to evaluate their biological activities.
Recently, a number of different quantum mechanical
theories have been advanced, for example, the density
functional theory (DFT). It is a computational quan-
tum mechanical modeling method used for studying
organic compounds. The DFT method is used as a
descriptor of chemical reactivity, orbitals, and the
energy bandgap.28b By considering the above facts
and their increasing importance in the pharmaceutical
and biological fields, and in continuation of our
program,29–31 this work has been devoted to develop-
ing new classes of heterocyclic systems that incorpo-
rate the isoxazole nucleus to explore their potential
antitumor activity.
with five membered rings containing two heteroatoms,
among which is isoxazole. It is an important heterocy-
clic unit that has been widely used as a key building
block for the synthesis of biologically active com-
pounds. The isoxazole nucleus and its derivatives are
attracting much attention in the fields of pharmacol-
ogy and medicine1,2 because of their various biological
functions,
including
antitumor,3
anti-HIV,4
antifungal,5 anticonvulsant,6 analgesic,7 herbicidal,8
and cestoidal activities.9 Isoxazole derivatives are also
employed in the treatment of leprosy10 and
diabetes,11–14 and have useful activities in conditions
like schizophrenia, hypertension, and Alzheimer’s dis-
ease, and in leflunomide (a disease-modifying anti-
rheumatic drug, DMARD), valdecoxib (a COX-2
inhibitor), and zonisamide (an anticonvulsant). The
active metabolite of leflunomide, A771726, is known
to inhibit the tumor necrosis factor (TNF)-α and inter-
leukin (IL)-1α from Kupffer cells in vitro.15 In addi-
tion, leflunomide is used to inhibit the production of
TNF-α, IL-1α, and IL-6 induced by bacterial lipopoly-
saccharides (LPSs) in peritoneal macrophages in rats
*Corresponding author. Email: wshamama@yahoo.com
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Hamama et al.
RESULTS AND DISCUSSION
Chemistry
5-Amino-3-methylisoxazole (1) was reacted with
chloroacetylchloride, furnishing the corresponding 2-
chloro-N-(3-methylisoxazol-5-yl)acetamide (2) by
NHCO proton. The MS spectrum showed a molecular
ion peak at m/z 315 (M+, 28.0%) and a base peak at
151. Furthermore, the signals that appeared in 13C
NMR of compound 4 are quite compatible with its
structure. The postulated mechanism for the formation
of compound 3 is shown in Scheme 2.
Furthermore, 2-cyano-N-(3-methyl-isoxazol-5-yl)
acetamide (5) was synthesized by treating 2 with potas-
sium cyanide in dimethylformamide (Scheme 3). Its IR
spectrum exhibits bands at 3494 cm−1 (NH), at
2229 cm−1 for (CN), and at 1727 cm−1 for (C=O). Its
1H NMR spectrum showed singlet signals at δ at 3.64
for (CH2) and at 9.70 ppm for NH. The MS spectrum
showed a molecular ion peak at m/z 165 (M+, 22.4%)
and a base peak at m/z 80.
a
modified procedure of Uyeo Shojiro et al. (Scheme 1).32
Compound 2 was treated with ammonium thiocyanate
in refluxing ethanol, affording 2-(3-methylisoxazol-5-
ylimino)thiazolidin-4-one (3). Compound 3 was synthe-
sized through intramolecular cyclization and the
Dimroth-like rearrangements of 3// under appropriate
reaction conditions.33,34 Its IR spectrum showed bands
at 3126 and 1724 cm−1 due to (N–H) and (C=O
amide), respectively. The lactam structure of 3 was con-
firmed on the basis of its 1H NMR spectrum, which dis-
played a singlet signal at δ 4.00 ppm assignable to CH2
protons and another singlet signal at δ 11.50 ppm of
the NHCO proton. The mass spectrum of 3 showed a
molecular ion peak at m/z 197 (M+, 82.3%) and a base
peak at m/z 82. It has been reported that the insertion
of arylidene moieties at the thiazolidin-4-one ring
enhanced its biological activity.35–37 Prompted by these
findings and as part of our efforts to discover poten-
tially active new antitumor agents, we report here the
condensation of 3 with p-anisaldehye in acetic acid and
sodium acetate to give 5-(4-methoxybenzylidene)-2-(3-
In addition to increasing the potent activity of 2-
chloro-N-(3-methylisoxazol-5-yl)acetamide (2) as an
anticancer agent,38,39 we built upon it other heterocylic
moieties of potential activity via the transformation of
2 with different heterocyclic systems containing nucleo-
philic centers to afford binary heterocycles. Therefore, the
alkylation of 2with 5-amino-1,3,4-thiadiazole-2-thiol (6)
in dry acetone gave α-(5-amino-1,3,4-thiadiazol-2-ylthio)-
N-(3-methylisoxazol-5-yl)acetamide (7). Its IR spectrum
showed bands at 3407, 3267, and 3164 cm−1for (NH) and
1
(NH2) and at 1655 cm−1 for (C=O). Its H NMR spec-
1
methylisoxazol-5-ylimino)thiazolidin-4-one (4). The H
trum showed five singlet signals corresponding to CH3,
CH2, isoxazole-CH, NH2, and NH at δ 2.17, 4.02, 6.11,
7.40, and 11.81 ppm, respectively. Its MS indicated a
molecular ion peak at m/z 271 (M+, 0.9%) and a base
peak at m/z 40.
NMR spectrum of 4 exhibits singlet signals for three
protons at δ 3.80 corresponding to the (OCH3) group, a
doublet at 7.08 (J = 8.34 Hz) for two aromatic protons,
a
doublet at 7.85 for two aromatic protons
(J = 8.33 Hz) and at 7.68 corresponding to ethylenic
proton, and a singlet signal at δ 11.50 ppm for the
In a similar manner, when 2 was reacted with 1H–
benzo[d]imidazole-2-thiol (8) in ethanol and triethylamine,
H3C
H3C
CH3
S
O
O
O
Cl
N
N
N
N
ClCCH2Cl
CHCl
NH4SCN
N
NH2
O
O
N
H
O
H
,
EtOH
reflux
1
,
N
3
2
3
H3CO
CHO
AcONa, AcOH
H3CO
CH3
S
O
HN
N
O
N
4
Scheme 1. Synthesis of thiazolidinone derivatves 3 and 4.
2
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JOURNAL OF THE CHINESE
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Synthesis Some New Isoxazole compounds
H3C
N
H3C
O
H3C
H3C
O
S
O
NH
NH4SCN
EtOH
N
O
S
..
NH
O
Cl
O
2
N
N
N
NH
N
O
O
NH
3//
N
C
S
3
Scheme 2. Plausible mechanism of the formation of compound 3.
H3C
Coumarins are important heterocyclic compounds
with great applications in medicinal chemistry; this core
structure can be found in compounds with diverse bio-
logical and medicinal applications.40 In this context, we
investigated the reaction of 5-amino-3-methylisoxazole
(1) as a 1,3-binucleophile with electrophilic compounds
such as 3-bromoacetylcoumarin 12 in ethanol under
reflux, which furnished 3-(3-methyl-6H-pyrrolo[3,2-d]
isoxazol-4-yl)-2H–chromen-2-one (13). Formation of
compound 13 is assumed to proceed via nucleophilic
displacement of the bromide to give an N-alkylated
intermediate, which subsequently undergoes tautomer-
ism with the elimination of water. IR spectrum of 13
showed bands at 3412 cm−1 for NH and at 1720 cm−1
O
KCN, DMF
N N
CN
S
N
N
H
O
5
H3C
O
HS
NH2
S
N
6
N
NH
N
O
O
K2CO3 , acetone
S
7
NH2
N
2
H3C
SH
N
O
H
S
8
N
N
N
H
NH
Ethanol
9
O
1
H3C
N
for C=O. In the H NMR spectrum of 13, three singlet
CH3
N
signals appeared at 5.52, 6.51, and 8.60 ppm for NH,
CH-pyrole, and CH-coumarin, respectively. Its MS
spectrum showed good agreement with its structure,
with a molecular ion peak at m/z 267 (M+ + H, 1.8%)
and a base peak at m/z 78.
O
N
NH2
H2N
H3C
O
CH3
O
10
N
O
N
CH3
DMF
N
H
O
11
Scheme 3. Synthesis of bioactive isoxazoles 5, 7, 9,
and 11.
Furthermore, continuation of our work on the syn-
thesis of biologically interesting heterocyclic compounds
containing the fused azepine heterocyclic ring, synthesized
by refluxing 1 with 2-(benzo[d]thiazol-2-yl)-4-chloro-3-
oxobutyronitrile (14) in DMF and triethylamine
(Scheme 4), led to the formation of 4-amino-5-(benzo[d]
thiazol-2-yl)-3-methyl-7,8-dihydro-6H-isoxazolo[5,4-b]aze-
pin-6-one (15). Formation of compound 15 is assumed to
proceed via the displacement of the chlorine of 14 with the
amino group of isoxazole followed by the attack of C-4 of
isoxazole, which is the more nucleophilic carbon to the
cyano group, affording 15. Its structure was elucidated by
it furnished 2-(1H–benzo[d]imidazol-2-ylthio)-N-(3-methyli-
soxazol-5-yl)acetamide (9) (Scheme 3). The IR spectrum
exhibits bands at 3201 and 3149 cm−1 for two (NH) and at
1
1691 cm−1 for (C=O). Its H NMR spectrum exhibits five
singlet signals corresponding to CH3, CH2, isoxazole-CH,
NH, and NH C=O at δ 2.27, 4.01, 6.21, 11.20, and 12.76,
respectively, and shows muliplets for four aromatic protons
at 6.93–7.83 ppm. Its MS shows a molecular ion peak at
m/z 288 (M+, 14%) and a base peak at m/z 200.
Also, 6-aminouracil has many medical and biolog-
ical activities. In the light of these, our study was
extended to the investigation of the reactivity of 2 with
6-aminouracil (10) in DMF, which gave 11. The 1H
NMR spectrum of compound 11 revealed three singlet
signals at 2.13 (CH3 isoxazole) and 3.2 and 3.4 (for two
CH3 pyrimidines), and singlet signals at 2.81 and
11.05 ppm for CH2 and NHCO protons, respectively.
1
IR and H NMR (see the Experimental section).The car-
bon skeleton of compound 15 was assigned using the 13C
NMR spectrum. Finally, the reaction of 1 with 1-chloro-
4-(chloromethyl)benzene (16) in methanol led to the for-
mation of 5-N-(4-chlorobenzyl)amino-3-methylisoxazole
1
(17). The H NMR spectrum of compound 17 displayed
two singlet signals at 4.60 and 4.80 for CH2 and NH ppm,
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Hamama et al.
Scheme 4. Alkylation of 5-amino-3-methylisoxazole (1).
respectively, and also two doublets at 7.34 (2H, d) and
7.81 (2H, d), which were readily assigned to the aromatic
protons. Its mass spectrum showed a molecular ion peak
at m/z 222 (M+, 14.8%) and a base peak at m/z 149.
The structures of all the newly synthesized com-
pounds were established on the basis of their microana-
lytical data as shown in the experimental section.
established tumor cell model Ehrlich ascite cells (EACs)
in vitro.41–43 The ED100, ED50, ED25, and IC50 values
of the active compounds are summarized in Table 1.
The data show clearly that compounds 7, 9, and 11 have
the highest cytotoxic activity in comparison to the well-
known cytotoxic antitumor agents such as 5-fluorouracil
(5-FU). However, compounds 13 and 17 were only mod-
erately potent compared to the other compounds. But
compounds 3, 4, and 15 showed low activities.
BIOLOGICAL ACTIVIY
Antitumor testing
Eight of the new synthesized isoxazole derivatives
were tested for cytotoxicity against well-known
Structure–activity relationship of antitumor activities
By comparing the results obtained for the investi-
gated compounds with their structures, the following
Table 1. In vitro cytotoxicity of isoxazole derivatives (Ehrlich ascites cells dead %)
% Death
Compound no.
IC50 μg
ED100 μg/mL
ED50 μg/mL
ED25 μg/mL
5-FU
3
4
7
9
11
13
15
17
32.21
74.21
80.43
11.52
30.53
8.54
52.65
98.43
48.32
96.1
22.0
17.0
96.3
96.4
97.6
70.2
38.0
80.5
67.0
13.3
11.4
65.7
68.4
68.3
39.0
17.0
41.4
42.9
6.5
6.1
41.5
44.8
44.2
28.8
10.0
21.5
IC50 is the inhibitive concentration. ED100, ED50, and ED25 are the effective doses at 25, 50, and 100 μL, respectively, of the
compounds used. Dead % refers to the percentage of dead tumor cells. 5-Fu is 5-fluorouracil, a well-known cytotoxic agent.
4
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Synthesis Some New Isoxazole compounds
Table 2. Binding energies and frontier orbital energies obtained from DFT
Compound
Binding energy (eV)
Total energy (eV)
HOMO (eV)
LUMO (eV)
E(HOMO – LUMO)
7 anti
9 anti
11 anti
−117.47872
−156.25778
−169.96766
−41 662.46368
−34 574.68075
−28 338.16501
−5.489
−5.582
−5.224
−1.854
−2.016
−1.558
−3.635
−3.566
−3.666
structure–activity relationships (SARs) were postulated:
(1) isoxazole derivatives 7, 9, and 11 were more potent
than 5-Fu, which may be attributed to the presence of
the 1,3,4-thiadiazole, benzo[d]imidazole, and pyrimidine-
2,4(1H,3H)-dione moieties, respectively; (2) compounds
13, 17, and 15 showed moderate activity, which may be
due to the presence of coumarin, p-chlorophenyl and aze-
pine moieties, respectively; and (3) finally, isoxazole deri-
vatives 3 and 4 had low activity compared to that of the
other compounds.
because they easily offer electrons to an acceptor. Also,
the low values of the energy gap may be due to the
groups or moieties entering those molecules. Therefore,
the energy gaps for the three compounds 7, 9, and 11
(Figures 1–3) with the highest antitumor activity were
determined. The results, indicated in Table 2 show that
compound 11 has the lowest energy gap, which means
the ease of charge transfer in 11, which enhanced its
biological activity compared to compounds 7 and 9.
Also, the isolated compound 11 showed large values of
binding energy than compounds 7 and 9, which
improved the stability of 11.46 Moreover, the energy
MOLECULAR MODELING
Geometry optimization
difference between the frontier orbitals (EHOMO
–
ELUMO) was found to be higher in the negative value in
the case of compound 7 than in compound 9, so the for-
mer’s cytotoxic activity as antitumor agent is higher
than that of compound 9.
Geometry optimization of the molecular structures
of the different compounds was carried out using DFT
via cluster calculations using the DMOL3 program44 in
the Materials Studio package45 in order to investigate
their stability relative to each other. The values
obtained for the main quantum parameters are sum-
marized in Table 2. According to the frontier molecular
orbital theory, chemical reactivity is a function of the
interaction between the HOMO and LUMO levels of
the reacting species.24b HOMO represents the ability to
donate an electron, whereas LUMO as an electron
acceptor represents the ability to obtain an electron.
The energy gap between EHOMO and ELUMO of the
molecules is an important parameter as a function of
the reactivity of molecules. Moreover, the experimental
results for the newly synthesized compounds were in
agreement with the theoretical data. By determining the
energy gap (EHOMO – ELUMO), one gets an indication
of the biological activity. Molecules with a small energy
gap are more polarized and reactive than hard ones
CONCLUSIONS
The objective of the present study was to synthesize
and investigate the antitumor activity of some novel iso-
xazole skeletons bearing other heterocyclic moieties,
starting from 5-amino-3-methylisoxazole. The data
showed that compounds 7, 9, and 11 are the most potent
compounds with higher cytotoxic activity than even
5-FU. The data reported here may be a helpful guide for
medicinal chemists who are working in this area.
EXPERIMENTAL
Materials and methods
All melting points are in Celcius (uncorrected)
and were determined by a Gallenkamp electric melting
point apparatus. Elemental analyses were carried out
Compound 9
9 HOMO
9 LUMO
Fig. 1. Molecular geometry and calculated HOMO and LUMO of compound 9 anti.
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Hamama et al.
Compound 7
7 HOMO
7 LUMO
Fig. 2. Molecular geometry and calculated HOMO and LUMO of compound 7 anti.
Compound 11
11 HOMO
11 LUMO
Fig. 3. Molecular geometry and calculated HOMO and LUMO of compound 11 anti.
at the Micro analytical Center, Faculty of Science,
Cairo University. IR spectra were recorded (in KB,
cm−1) on a Mattson 5000 FTIR spectrophotometer at
the Micro analytical Center, Faculty of Science, Man-
(10 mL) containing pyridine (0.49 g, 5 mmol) was
added choloracetyl chloride (0.56 g, 5 mmol), with the
temperature maintained at 0–10ꢀC during addition. The
reaction mixture was stirred for 1 h and then poured
into cold water. The formed precipitate was filtered off,
washed with water, and recrystallized from ethanol to
give 2. White crystals; 99% yield; mp 131–132ꢀC (etha-
nol) (Lit. mp [32] =130–132ꢀC); Rf = 0.5 [pet. Ether
(40–60)/ethyl acetate (1:3)].
1
soura University. H NMR spectra were measured on
a Varian spectrometer at 400 MHz, using TMS as
internal reference and DMSO-d6 or CDCl3 as solvent
at the National Research Center, Cairo. 13C NMR
Spectra were acquired on a JEOL ECX-400 spectrome-
ter operating at 100 MHz at room temperature in
CDCl3 and DMSO-d6 using a 5 mm probe at the
Chemistry department, School of Engineering and Sci-
ence, University of Jacobs, Bremen, Germany. The
chemical shifts (δ) are reported in parts per million and
referenced to the residual solvent peak. Mass spectra
were recorded on a Kratos (70 eV) MS instrument
and/or a Varian MAT 311 mass spectrometer at Micro
analytical Center, Cairo University. Reaction mixtures
were monitored by thin-layer chromatography (TLC)
using EM science silica gel-coated plates with visualiza-
tion by irradiation with an ultraviolet lamp. Fetal
bovine serum was from GIBCO, UK, and the RPMI-
1640 medium from Sigma chemical Co. (St. Louis,
MO, USA). Biological testing was carried out at Drug
Department, Faculty of Pharmacy, Mansoura Univer-
sity, Mansoura, Egypt. Ehrlich cells (Ehrlich ascites
carcinoma, EAC) were derived from the ascetic fluid of
diseased mice (the cells were purchased from the
National Cancer institute, Cairo, Egypt).
(Z)-2-(3-Methylisoxazol-5-ylimino)thiazolidin-4-
one (3). A mixture of 2-chloro-N-(3-methylisoxazol-5-
yl)acetamide (2) (0.87 g, 5 mmol) and ammonium thi-
ocyanate (0.76 g, 0.1 mol) in absolute ethanol (50 mL)
was refluxed for 5 h, and then the reaction mixture
was allowed to stand overnight at room temperature.
The precipitate formed was filtered off, washed with
water, and recrystallized from ethanol to give 3. White
crystals; 80% yield; mp 255–257ꢀC; Rf = 0.3 [pet.
Ether (40–60)/ethyl acetate (1:3)]; IR (cm−1): 3126
1
(NH), 1724 (CO), 1624 (C=N); H NMR (DMSO-d6)
δ: 2.31 (s, 3H, CH3), 4.00 (s, 2H, CH2), 6.11 (s, 1H,
isoxazole-CH), 11.50 (s, 1H, NHCO);MS: (EI, 70 eV)
m/z (%) = 199 (M+ + 2, 0.5), 198 (M+ + H, 4.7),
197 (M+, 82.3), 143 (9.2), 142 (2.7), 124 (36.9),
115 (8.5), 98 (4.4), 97 (2.3), 82 (100.0, base peak),
69 (29.6), 68 (10.8); Anal. Calcd. for C7H7N3O2S
(197.21): C, 42.63; H, 3.58; N, 21.31; Found: C,
42.65; H, 3.65; N, 21.29.
(2Z,5E)-5-(4-Methoxybenzylidene)-2-(3-methyli-
2-Chloro-N-(3-methylisoxazol-5-yl)acetamide (2).
To a solution of 1 (0.49 g, 5 mmol) in chloroform
soxazol-5-ylimino)thiazolidin-4-one (4). A mixture of
3 (0.79 g, 4 mmol) in acetic acid (35 mL) was buffered
6
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Synthesis Some New Isoxazole compounds
with sodium acetate (8 mmol) followed by the addition
of anisaldehyde (0.73 mL, 6 mmol). The solution was
refluxed for 2 h, then allowed to stand overnight, and
the precipitate formed was filtered off and washed with
ethanol. The resulting crude product was purified by
recrystallization from ethanol tofurnish 4: Yellow crys-
tals; 85% yield; mp 225–227ꢀC; Rf = 0.3 [pet. Ether
(40–60)/ethyl acetate (1:3)]; IR (cm−1): 3120 (NH),
3056, 2966 (CH, str.), 1724 (C=O), 1644 (C=N), 1616
2-(5-Amino-1,3,4-thiadiazol-2-ylthio)-N-(3-methyli-
soxazol-5-yl) acetamide (7). A mixture of 2 (0.56 g,
4 mmol), anhydrous potassium carbonate (0.55 g,
4 mmol), and 5-amino-1,3,4-thiadiazole-2-thiol (6)
(0.532 g, 4 mmol) in dry acetone (20 mL) was heated
under reflux for 6 h, and then the reaction mixture was
allowed to stand at room temperature overnight. The
formed precipitate was collected by filtration, washed
with ethanol, and recrystallized from benzene to fur-
nished 7: White crystals; 60% yield; mp 215–217ꢀC;
Rf = 0.3 [pet. Ether (40–60)/ethyl acetate (1:3)]; IR
(cm−1): (3407, 3267 (NH2), 3164 (NH), 1655 (C=O),
1645, 1608 (C=N), 1217, 1062 (C–S–C); 1H NMR
(DMSO-d6) δ: 2.17 (s, 3H, CH3), 4.02 (s, 2H, CH2),
6.11 (s, 1H, isoxazole-CH), 7.90 (s, 2H, NH2), 11.81 (s,
1H, NH); MS: (EI, 70 eV) m/z (%) = 272 (M++ H,
0.53), 271 (M+, 0.9), 270 (M+– 1, 4.1), 174 (85.67),
100 (12.3), 98 (55.0), 97 (14.8), 82 (57.2), 40 (100.0, base
peak); Anal. Calcd. for C8H9N5O2S2 (271.31): C,
35.41; H, 3.34;N, 25.81; Found: C, 35.39; H,
3.33; N, 25.78.
1
(C=C); H NMR (DMSO-d6) δ: 2.19 (s, 3H, CH3), 3.80
(s, 3H, OCH3), 6.03 (s, 1H, isoxazole-CH), 7.08 (d,
J = 8.34 Hz, 2H, aromatic-CH), 7.85 (d, J = 8.33 Hz,
2H, aromatic-CH), 7.68 (s, 1H, CH = C), 11.50 (s, 1H,
NHCO); 13C NMR (DMSO-d6), δ: 12.22 (CH3), 56.03
(CH3-O), 93.54 (CH-isoxazole), 115.52, 115.90, 119.69,
121.98, 125.98, 132.27 (C=C), 132.77 (CH = C), 156.95
(C-N), 161.75 (C-O), 161.73 (C=N), 167.41 (N = C-
CH3), 168.16 (C=O); MS: (EI, 70 eV) m/z (%) = 315
(M+, 28.0), 314 (M+– 1, 14.4), 257 (26.2), 241 (17.6),
211 (37.1), 197 (16.7), 174 (19.9), 151 (100.0, base
peak), 137 (16.4), 105 (24.4), 78 (24.6); Anal. Calcd. for
C15H13N3O3S (315.35): C, 57.13; H, 4.16;N, 13.33;
Found: 57.15; H, 4.17; N, 13.34.
2-(1H–Benzo[d]imidazol-2-ylthio)-N-(3-methylisox-
azol-5-yl)acetamide (9). A mixture of 2 (0.56 g, 4 mmol)
and 1H–benzo[d]imidazole-2-thiol (8) (0.4 g, 4 mmol)
in dioxane (20 mL) was heated under reflux for 3 h,
and then the reaction mixture was allowed to stand at
room temperature overnight. The formed precipitate
was collected by filtration, washed with ethanol, and
recrystallized from ethanol to give 9: Black crystals;
90% yield; mp 180–181ꢀC; Rf = 0.5 [pet. Ether (40–60)/
ethyl acetate (3:4)]; IR: (cm−1) 3201, 3149 (2NH), 2908,
2-Cyano-N-(3-methylisoxazol-5-yl)acetamide (5).
Compound 2 (0.56 g, 5 mmol) was dissolved in
dimethylformamide (5 mL), and to the resulting clear
solution was added potassium cyanide (0.36 g,
5.5 mmol) in water (1 mL) while stirring. Addition
was controlled so that the temperature of the reac-
tion mixture was maintained at 70ꢀC. After complete
addition of the cyanide solution, stirring was contin-
ued for a further 15 min. Then the reaction mixture
was cooled and added to an ice-cold solution of
conc. Hydrochloric acid (0.5 mL) in water (100 mL).
The precipitate formed was filtered off, washed with
water, dried, and recrystallized from ethanol to give
5. Yellow crystals; 80% yield; mp 120–121ꢀC; Rf =
0.3 [pet. Ether (40–60)/ethyl acetate (1:3)]; IR:
(cm−1) 3494 (NH), 2954, 2929 (CH, str.), 2229 (CN),
1
2815 (CH, str.), 1691 (C=O), 1616 (C=N); H NMR
(DMSO-d6) δ: 2.27 (s, 3H, CH3), 4.01 (s, 2H, CH2),
6.21 (s, 1H, isoxazole-CH), 6.93–7.83 (m, 4H,
Aromatic-CH), 11.20 (s, 1H, NH), 12.76 (s, 1H, NH
C=O); MS: (EI, 70 eV) m/z (%) = 288 (M+, 14),
216 (25.2), 200 (100.0, base peak), 199 (20.5), 102 (9.8),
90 (9.0);Anal. Calcd. for C13H12N4O2S (288.32): C,
54.16; H, 4.20; N, 19.43; Found: C, 54.15; H,
4.22; N, 19.40.
1
1727 (C=O), 1621 (C=N); H NMR (CDCl3) δ: 2.27
(s, 3H, CH3), 3.64 (s, 2H, CH2), 6.24 (s, 1H,
isoxazole-CH), 9.70 (s, 1H, NH); MS: (EI, 70 eV) m/
z (%) = 167 (M++ 2, 1.6), 165 (M+, 22.4), 149 (2.5),
129 (2.9), 123 (2.5), 115 (2.0), 91 (8.9), 80 (100.0,
base peak); Anal. Calcd. for C7H7N3O2 (165.15): C,
50.91; H, 4.27;N, 25.44; Found: C, 50.87; H,
4.25; N, 25.42.
2-(6-Amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahy-
dropyrimidin-5-yl)-N-(3-methylisoxazol-5-yl)acetamide
(11). A mixture of 2 (0.56 g, 4 mmol) and 6-amino-
1,3-dimethyl-pyrimidine-2,4-(1H,3H)-dione
(10)
(0.62 g, 4 mmol) in DMF (50 mL) was heated under
reflux for 3 h, and then the reaction mixture was
allowed to stand overnight. The precipitate formed
J. Chin. Chem. Soc. 2017
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7

Article
Hamama et al.
was collected by filtration, washed with ethanol, and
then recrystallized from ethanol to afford 11. Yellow
crystals; 70% yield; mp 220–222ꢀC; Rf = 0.2 [pet.
Ether (40–60): ethyl acetate (1:4)]; IR (cm−1): 3397,
3351 (NH2), 2996, 2950 (CH, str.), 1660 (C=O), 1610
2.30 (s, 3H, CH3), 4.54 (s, 2H, CH2), 6.43 (s, 2H,
NH2), 7.13–7.99 (m, 4H, aromatic-CH), 8.30 (s, 1H,
NH); 13CNMR (DMSO-d6) δ: 22.24 (CH3), 55.58,
113.14, 113.14, 114.79 (C=N), 121.39, 121.42, 124.55,
124.82, 127.08 (C–S), 128.78, 145.54 (C–N), 148.96,
167.56, and 191.20 (C=O); Anal. Calcd. for
C15H12N4O2S (312.35): C, 57.68; H, 3.87;N, 17.94;
Found: C, 57.70; H, 3.88; N, 17.95.
1
(C=C); H NMR (DMSO-d6) δ: 2.13 (s, 3H, CH3-iso-
xazole), 2.81(s, 2H, CH2), 3.2 (s, 3H, CH3 pyrimidine),
3.40 (s, 3H, CH3-pyrimidine), 6.12 (s, 1H, isoxazole-
CH), 6.90 (s, 2H, NH2), 11.05 (s, 1H, NHCO); MS:
(EI, 70 eV) m/z (%) = 295 (M++2, 1.1), 292 (M+ − H,
1.8), 211 (2.2), 155 (16.1), 82 (20.5), 44 (100.0, base
peak); Anal. Calcd. for C12H15N5O4 (293.28): C,
49.14; H, 5.16; N, 23.88; Found: C, 49.13; H,
5.15; N, 23.86.
N-(4-Chlorobenzyl)-3-methylisoxazol-5-amine (17).
A mixture of 1 (0.49 g, 5 mmol) and 1-chloro-4-(chloro-
methyl) benzene (16) (0.80 g, 5 mmol) in absolute etha-
nol (50 mL) was heated under reflux for 3 h, and then
the reaction mixture was allowed to stand overnight. The
precipitate formed was collected by filtration, washed
with ethanol, and then recrystallized from ethanol to
afford 17: Yellow crystals; 57% yield; mp > 300ꢀC; Rf =
0.5 [pet. Ether (40–60)/ethyl acetate (2:5)]; IR (cm−1):
3139 (NH), 2823 (CH, str.), 1533 (C–N) cm−1; 1H NMR
(CDCl3) δ: 2.10 (s, 3H, CH3), 4.60 (s, 2H, CH2N), 4.80
(s, 1H, NH), 6.31 (s, 1H, isoxazole-CH), 7.34 (d,
J = 9.01 Hz, 2H, aromatic-CH), 7.81 (d, J = 8.97 Hz,
2H, aromatic-CH); MS: (EI, 70 eV) m/z (%) = 223 (M+
+ 1, 6.6), 222 (M+, 14.8), 207 (8.6), 112 (56.6),
111 (9.07), 126 (20.5), 141 (18.2), 149 (100.0, base peak),
123 (44.5), 121 (31.7), 97 (12.9), 96 (24.0), 73 (21.1),
67 (65.8); Anal. Calcd. for C11H11ClN2O (222.67): C,
59.33; H, 4.98;N, 12.58; Found: C, 59.29; H,
4.97; N, 12.56.
3-(3-Methyl-6H-pyrrolo[3,2-d]isoxazol-4-yl)-2H–
chromen-2-one (13). A mixture of 1 (0.49 g, 5 mmol)
and 3-bromoacetyl coumarin (12) (1.33 g, 5 mmol) was
refluxed in ethanol (50 mL) for 5 h. The reaction mix-
ture was allowed to stand overnight at room tempera-
ture. The resultant solid (hydrobromide) was separated
by filtration. The free base was obtained by neutraliza-
tion with sodium bicarbonate solution to give the solid
product. It was washed with water and then recrystal-
lized from ethanol to give 13: Red crystals; 66% yield;
mp 161–162ꢀC (ethanol); Rf = 0.6 [pet. Ether (40–60):
ethyl acetate (1:3)]; IR (cm−1): 3412 (NH), 2970, 2925
1
(CH, str.), 1720 (CO), 1607 (C=C); H NMR (CDCl3)
δ: 2.11 (s, 2H, CH3), 5.52 (s, 1H, NH),6.51 (s, 1H, CH-
pyrole), 7.41–7.91 (m, 4H, aromatic-CH), 8.60 (s, 1H,
CH-coumarin); MS (EI, 70 eV) m/z (%) = 268 (M+
+
Antitumor activity using Ehrlich ascites in vitro assay
Different concentrations of the tested compounds
were prepared (100, 50, and 25 μg/mL, DMSO). Asci-
tes fluid from the peritoneal cavity of the donor ani-
mal (containing Ehrlich cells) was aseptically
aspirated. The cells were grown partly floating and
partly attached in a suspension culture in RPMI 1640
medium, supplemented with 10% fetal bovine serum.
They were maintained at 37ꢀC in a humidified atmos-
phere with 5% CO2 for 2 h. The viability of the cells
was determined by the microscopic examination using
a hemocytometer and using trypan blue stain (stains
only the dead cells).47,48
2, 3.9), 267 (M+ + 1, 1.8), 205 (2.6), 173 (15.3),
168 (1.3), 145 (3.9), 121 (80.0), 80 (16.6), 79 (38.4),
78 (100.0, base peak); Anal. Calcd. for C15H10N2O3
(266.25): C, 67.67; H, 3.79; N, 10.52; Found: C,
67.64; H, 3.78; 10.54.
4-Amino-5-(benzo[d]thiazol-2-yl)-3-methyl-7,8-dihy-
dro-6H-isoxazolo [5,4-b]azepin-6-one (15). A mixture of
1
(0.49 g, 5 mmol) and 2-(benzo[d]thiazol-2-yl)-4-
chloro-3-oxobutyronitrile (14) (1.25 g, 5 mmol) in
DMF (10 mL) and triethylamine (three drops) was
refluxed for 3 h. The reaction mixture was allowed to
stand overnight at room temperature to give brown
crystals, which was collected by filtration and recrystal-
lization from DMF to afford 15: Brown crystals; 60%
yield; mp 290–292ꢀC; Rf = 0.6 [pet. Ether (40–60)/ethyl
acetate (1:3)]; IR (cm−1): 3409, 3400 (NH2), 1695
ACKNOWLEDGMENTS
We deeply appreciate the assistance of Prof.
Dr. N. Kuhnert, Professor of Organic Chemistry,
Chemistry Department, School of Engineering and
(C=O), 1614 (C=N) cm−1 1H NMR (DMSO-d6) δ:
;
8
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© 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2017

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Synthesis Some New Isoxazole compounds
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Science, Jacobs University, Bremen, for carrying out
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MS) of some of our samples. This work was sup-
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